During antidiuresis, the osmolality in the inner medulla of the mammalian kidney increases up to 1,200 mosmol/kg H₂O in humans, thus allowing efficient urinary concentration. Renal medullary cells express osmoprotective genes involved in the accumulation of organic osmolytes, thereby restoring intracellular ionic strength to near-normal values. Although it is clearly established that TonEBP contributes to the expression of these genes, neither the precise signaling mechanism by which hypertonicity activates TonEBP is completely understood, nor is it known whether a membrane-bound osmosenser, corresponding to yeast and bacteria, is present in mammalian cells. We found evidence that metalloproteinase (MMP)-dependent activation of the EGF receptor (EGFR) signals to TonEBP and stimulates the expression of the TonEBP target gene aldose reductase (AR) under hypertonic conditions. Phosphorylation of EGFR and the downstream MAP kinases ERK1/2 and p38 were significantly enhanced by high NaCl in MDCK cells. Conversely, the broad-spectrum MMP inhibitor GM6001 or the EGFR inhibitor AG1478 diminished phosphorylation of EGFR, p38, and ERK1/2, the induction of AR mRNA and protein, and AR promoter reporter activity in response to hypertonicity. Accordingly, neutralizing antibodies against the putative EGFR ligand transforming growth factor-a (TGF-a) abolished AR induction during osmotic stress. Furthermore, tonicity-induced phosphorylation of p38 and ERK1/2, and expression of AR was reduced significantly in MDCK cells transfected with a dominant-negative Ras construct. These effects were not caused by reduced nuclear abundance of TonEBP during osmotic stress, suggesting that EGFR signaling is not involved in nuclear translocation of TonEBP. However, TonEBP transactivation activity was diminished under hypertonic conditions in the presence of AG1478 and GM6001. The contribution of MMP/EGFR signaling in vivo was confirmed in C57BL/6 mice, in which treatment with GM6001 was associated with reduced AR induction following dehydration. Taken together, these results indicate, that osmotic stress induces MMP-dependent activation of EGFR, likely via shedding of TGF-a, and downstream-activation of Ras and the MAP kinases p38 and ERK1/2, which stimulate TonEBP transactivation activity. This EGFR-Ras-MAPK pathway contributes to TonEBP transcriptional activation and target gene expression during osmotic stress, thus establishing a membrane-associated signal input that contributes to the regulation of TonEBP activity.